The invention relates to a purified form of stabilized thrombin-activatable fibrinolysis inhibitor (TAFI), a method of producing stabilized TAFI, methods for therapeutic, prophylactic and diagnostic use of stabilized TAFI compositions and inhibitors thereof and kits comprising stabilized TAFI useful in measuring TAFI and other carboxypepetidase activity using the stabilized TAFI compositions of the invention as a standard.
A proper balance between the activities of coagulation and fibrinolytic cascades is needed both to protect an organism from excessive blood loss upon injury and to maintain blood flow within the vascular system. The two opposing coagulation and fibrinolytic cascades are recognized to comprise a series of zymogen to enzyme conversions which terminate in the two respective proteolytic enzymes, thrombin and plasmin. These enzymes catalyze the formation and removal of fibrin within the circulatory system. Imbalances are characterized by either bleeding or thrombotic tendencies which may result in heart attacks or strokes in the organism.
Thrombin activatable fibrinolytic inhibitor (TAFI) is a 60 KDa glycoprotein present in human plasma that modulates fibrinolyisis in vivo. TAFI present in plasma is a proenzyme form which is most efficiently activated by proteolytic cleavage at Arg-92 with a thrombin-thrombomodulin complex. The proenzyme form of TAFI may also be activated by proteolytic cleavage by other proteolytic enzymes including, but not limited to, thrombin or plasmin. Upon activation of the TAFI proenzyme by proteolytic cleavage with thrombin-thrombomodulin, an active enzyme of 35 KDa is formed with carboxypeptidase-like activity (TAFIa). This molecule has also been referred to in the literature as plasma procarboxypeptidase B (PCPB), or plasma carboxypeptidase U (PCPU).
Modulation of fibrinolysis occurs when TAFIa cleaves C-terminal arginine and lysine residues of partially degraded fibrin, thereby inhibiting the stimulation of tissue plasminogen activator (t-PA) modulated plasminogen activation. The fibrinolytic system is activated primarily by t-PA which is provided by damaged cells in the blood vessel wall. t-PA converts circulating plasminogen to the active protease plasmin and can produce either slow enhancement of fibrinolysis or, when combined with fibrin, rapid enhancement of fibrinolysis. The effect of t-PA on fibrinolysis can be blocked by a class of inhibitors termed plasminogen activator inhibitors (PAIs), of which several have been identified.
Thrombomodulin is a component of the blood vessel wall which binds thrombin and changes its specificity from fibrinogen to protein C, resulting in a molecule possessing anticoagulant, rather than procoagulant, activity. The thrombin-thrombomodulin complex catalyzes cleavage of protein C to activated protein C, which results in down-regulation of the coagulation cascade by proteolytically inactivating the essential cofactors, Factor Va and VIIIa. In this manner, the body regulates coagulation cascade.
Studies such as that by Taylor et al., Thromb. Res. 37:639 (1985) have suggested that activated protein C is not only an anticoagulant, but also profibrinolytic, both in vivo and in vitro. Subsequently, it was determined that protein C only appears profibrinolytic because it prevents the thrombin-catalyzed activation of a previously unknown fibrinolysis inhibitor, whose precursor was isolated from plasma and designated as being TAFI.
TAFI was discovered independently in three different laboratories. In initially appeared as an unstable carboxypeptidase B-like molecule in human serum and was described by Hendriks et al., Biochim. Biophys. Acta 1034:86 (1990). A year later the cDNA for the molecule was cloned, its amino acid sequence was described, its activation by trypsin and its enzymatic properties toward synthetic carboxypeptidase B substrates was reported (see U.S. Pat. No. 5,206,161). In 1994, Wang et al., (J. Biol. Chem. 269:15937 (1994)) isolated the activated molecule and named it carboxypeptidase U (xe2x80x9cUxe2x80x9d being designated for unstable). Subsequently, Nesheim et al. (J Biol. Chem. 270:14477 (1995)) showed that the protein was both activated by thrombin and inhibits fibrinolysis, and designated the molecule TAFI. The co-identity of PCPB, PCPU, and TAFI has been established by their independent chromatographic behavior on plasminogen Sepharose(copyright) and the amino acid sequences present at the activation cleavage site.
The mechanism of TAFI inhibition of fibrinolysis can be schematically described as depicted in FIG. 1.
Diagnostic assays for TAFIa may be useful in the prognosis or diagnosis of certain hemorrhagic or thrombotic diseases or disorders. Accordingly, there is a need in the art for effective carboxypeptidase activity assays.
As TAFIa is believed to play a central regulatory role in the fibrinolytic cascade, the manipulation of TAFIa levels or activity in biological fluids may have important therapeutic applications with respect to hemorrhagic disorders including, but not limited to, vascular and heart pathologies, and stroke. Inhibitors of TAFIa would enhance fibrinolysis and have an anti-coagulant effect (see U.S. Pat. No. 5,993,815). Inhibitors of TAFIa could also be effective at treating or preventing the inflammation associated with arthritis as vascular endothelial growth factor (VEGF) is a potential substrate of TAFIa. VEGF has been linked with arthritis (Farva, R. A., J. Exp. Med. 180:341-6 (1994)).
Conversely, due to its ability to inhibit fibrinolysis, TAFIa itself may be useful as a therapeutic protein capable of acting as a procoagulant. In hemophilia A, a disease characterized by excessive bleeding, the addition of a protein mixture containing TAFIa to hemophilia A plasma has been reported to increase plasma clot lysis times (Mosnier, L. O. et al., Thromb. Haemost. 86:1035-9 (2001)). Additionally, thrombolytic therapy with tissue plasminogen activator (t-PA) for acute ischemic stroke remains complicated by risks of hemorrhagic transformation (Sumii T. and Lo E. H., Stroke 33:831-6 (2002)).
Unfortunately, the use of TAFIa as a therapeutic protein has not been feasible due to the inherent instability of the enzyme which has a half-life of less than 10 minutes at 37xc2x0 C. Consequently, TAFIa is also extremely difficult to purify due to its highly unstable nature. Thus, there remains a need in the art for a method of producing a more thermally stable form of TAFIa, including purified forms of stabilized TAFIa, which could have widespread diagnostic use as well as therapeutic use in the treatment, prevention or management of hemorrhagic diseases or disorders.
Citation or identification of any reference in this section is not to be construed as such reference being prior art to the present application.
The present invention is based in part on the surprising discovery that TAFIa can be prepared in a stabilized form by activation of TAFI by proteolytic cleavage in a substantially calcium-free environment. In brief, the present invention is related to a method of preparing a stabilized form of TAFIa, methods for diagnostic and/or therapeutic use of stabilized TAFIa, methods of therapeutic use of TAFIa inhibitors, and kits comprising stabilized TAFIa useful in measuring carboxypepetidase activity, particularly TAFI activity.
In one embodiment, the present invention encompasses a method of producing a stabilized form of TAFIa, or a pharmaceutical composition thereof. In particular, the method of producing stabilized TAFIa comprises protease catalyzed activation of TAFI to TAFIa in a substantially calcium-free environment. In one embodiment, the proteolytic cleavage is catalyzed by thrombin. In another embodiment, the proteolytic cleavage is catalyzed by plasmin. In another embodiment, the proteolytic cleavage is catalyzed by a thrombin-thrombomodulin complex.
In one embodiment, the TAFIa is prepared from wild-type TAFI (e.g., purified from a native or recombinant source). The wild-type TAFI can be naturally occurring or recombinant. In another embodiment, the TAFIa is prepared from a mutant, variant, analog, polymorphism or derivative of TAFI, preferably where the mutant, variant, analog, polymorphism or derivative of TAFI can be processed to TAFIa that exhibits one or more activities of TAFIa. The mutant, variant, analog or derivative of TAFI can be naturally occurring or recombinantly produced.
In certain embodiments, stabilized TAFIa is then purified from remaining TAFI and other components of the activation reaction.
In another embodiment, the present invention is directed to a method of treating, preventing, managing or ameliorating the symptoms of a hemorrhagic disease or disorder comprising administering a therapeutically or prophylactically effective amount of stabilized TAFIa, or a pharmaceutical composition thereof, to a patient having such a disease or disorder. In certain embodiments the hemorrhagic disease or disorder includes, but is not limited to, hemophilia, von Willebrand disease (VWD), Henoch-Schonlein purpura, Factor deficiencies and hemostasis.
In another embodiment, the present invention is directed to a method of treating, preventing or managing bleeding side-effects associated with the administration of tissue plasminogen activator (t-PA), or an analog thereof, aspirin (particularly overdose) and other anti-coagulants, comprising administering a therapeutically or prophylactically effective amount of stabilized TAFIa, or a pharmaceutical composition thereof, to a patient having such a disease or disorder. In certain embodiments the bleeding side-effects include, but are not limited to, hemorrhagic transformation (HT).
In another embodiment, the present invention is directed to a method of treating, preventing or managing thrombotic diseases or disorders comprising administering a therapeutically or prophylactically effective amount of an inhibitor of TAFIa, or a pharmaceutically acceptable composition thereof, to a patient having such a disease or disorder. Inhibitors of TAFIa include, but are not limited to, captopril, captopril-phe, pro-phe-argininic acid, pro-trp-argininic acid, pro-tyr-argininic acid, pro-phe, pro-trp, pro-tyr and argininic acid. In certain embodiments the thrombotic diseases or disorders include, but are not limited to, heart attack, stroke, thromboembolic disease, acute myocardial infarction (AMI), deep vein thrombosis, acute ischemic stroke, massive pulmonary embolism, disseminated intravascular coagulation (DIC), anti-phospholipid syndrome, familial thrombophilia, sepsis, arthritis, fulminant hepatitis and thrombosis.
In another embodiment, the stabilized TAFIa, inhibitor of TAFIa, or pharmaceutical composition thereof, can be administered in combination with another prophylactic or therapeutic agent. In certain embodiments, the other therapeutic or prophylactic agent is useful for the treatment, prevention or management of a hemorrhagic or thrombotic disorder. In other embodiments, the therapeutic or prophylactic agent is not useful for the treatment, prevention or management of a hemorrhagic or thrombotic disorder. When used in combination with other prophylactic and/or therapeutic agents, the stabilized TAFIa, inhibitor of TAFIa, or pharmaceutical compositions thereof, can be administered prior to, subsequent to or concurrently with the other therapeutic or prophylactic agents.
In another embodiment, the present invention is directed to methods comprising the use of stabilized TAFIa as a standard in a diagnostic assay. In a preferred embodiment, the diagnostic assay allows for the determination of carboxypeptidase activity of a purified enzyme sample or biological samples such as, but not limited to, plasma, urine, serum, semen and cerebral or spinal fluid, by comparison to the activity of a stabilized TAFIa standard. In a more preferred embodiment, the diagnostic assay measures TAFIa activity of a purified enzyme sample or biological samples such as plasma. In a preferred embodiment, the carboxypeptidase activity of TAFIa is measured in a chromogenic assay.
In another embodiment, the invention is directed to a kit comprising the components of the chromogenic carboxypeptidase diagnostic assay. In a certain embodiment, the kit comprises a stabilized TAFIa standard; a specially formulated developer comprising a carboxypeptidase substrate such as hippuryl-L-lysine or the tetrapeptide pro-phe-gly-lys (PFGK); lysine oxidase; horseradish peroxidase (HRP); a chromogenic substrate of HRP, such as O-phenylene diamine (OPD) or TMB; in any appropriate buffer, e.g., 200 mM Tris-HCl, pH 7.4 buffer.
In another embodiment, the present invention is directed to a method of measuring carboxypeptidase activity in a fluorescent-based assay using stabilized TAFIa as a standard. In a preferred embodiment, the carboxypeptidase activity of TAFIa is measured in the fluorescent-based assay.
In another embodiment, the invention is directed to a kit comprising the components of the fluorescent-based diagnostic assay. In a certain embodiment, the kit comprises a stabilized TAFIa standard; a specially formulated developer comprising a carboxypeptidase substrate such as hippuryl-L-lysine or the tetrapeptide pro-phe-gly-lys (PFGK); lysine oxidase; horseradish peroxidase (HRP); a chromogenic substrate of HRP, such as O-phenylene diamine (OPD) or TMB; in any appropriate buffer, e.g., 200 mM Tris-HCl, pH 7.4 buffer.
In another embodiment, the present invention is directed a method of using the tetrapeptide pro-phe-gly-lys (PFGK), hippuryllysine or hippurylarginine as a substrate for TAFIa in a diagnostic carboxypeptidase activity assay.
These and other aspects of this invention will be evident upon reference to the following detailed description and figures. To that end, certain patent and other documents are cited herein to more specifically set forth various aspects of this invention. Each of these documents are hereby incorporated by reference herein in its entirety.
3.1 Definitions
The term xe2x80x9cactivexe2x80x9d as used to describe stabilized TAFIa means TAFIa with a Vmax preferably above about 10 xcexcmol/min, more preferably, above about 15 xcexcmol/min, more preferably above about 20 xcexcmol/min, and most preferably, above about 25 xcexcmol/min.
The terms xe2x80x9canalogxe2x80x9d, xe2x80x9cpolymorphismxe2x80x9d and xe2x80x9cvariantxe2x80x9d as used herein refers to a polypeptide that possesses a similar or identical function as a particular protein (e.g., TAFIa), or a fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence or structure of that protein complex or a fragment thereof. A polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of the protein or protein complex or a fragment thereof as described herein (e.g., human or murine TAFI or TAFIa, e.g., having an amino acid sequence of accession No. NPxe2x80x94001863 or NPxe2x80x94062749, respectively, or the mature protein thereof); (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a protein or protein complex of the invention, or fragment thereof, as described herein of at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence encoding the protein or protein complex of the invention or a fragment thereof as described herein. A polypeptide with similar structure to a protein or protein complex of the invention or a fragment thereof as described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of said protein or protein complex or a fragment thereof as described herein. The structure of a polypeptide can be determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.
The term xe2x80x9cderivativexe2x80x9d as used herein refers to a polypeptide that comprises an amino acid sequence of a protein (e.g., TAFIa) or protein complex of the invention or a fragment thereof as described herein that has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term xe2x80x9cderivativexe2x80x9d as used herein also refers to a protein or protein complex of the invention or a fragment thereof that has been modified, i.e., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a protein or protein complex or a fragment thereof may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of a protein or protein complex or a fragment thereof may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a protein or protein complex or a fragment thereof may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a protein or protein complex or a fragment thereof described herein.
The phrase xe2x80x9cfree calciumxe2x80x9d means calcium that is not bound, chelated or otherwise sequestered.
The phrase xe2x80x9cmodulation of fibrinolysisxe2x80x9d means the inhibition or activation of the fibrinolytic cascade which may have an anti-coagulant or procoagulant effect.
The phrases xe2x80x9csubstantially calcium-free environmentxe2x80x9d or xe2x80x9csubstantially free from calciumxe2x80x9d mean a free calcium concentration (i.e., not chelated or otherwise sequestered) of less than 100 xcexcM, more preferably less than 10 xcexcM, more preferably less than 1 xcexcM, more preferably less than 500 nM, more preferably less than 100 nM , more preferably less than 1 nM, more preferably less than 100 pM, and most preferably less than 1 pM.
xe2x80x9cTAFIa inhibitorxe2x80x9d or xe2x80x9cinhibitors of TAFIaxe2x80x9d means any molecule that blocks, reduces or retards the cleavage of fibrin or other substrates by TAFIa.
The phrase xe2x80x9cstabilized TAFIaxe2x80x9d means TAFIa that has a half-life of at least one hour at room temperature, more preferably of at least two hours, more preferably of at least four hours, more preferably of at least eight hours, more preferably of at least twelve hours, more preferably of at least twenty-four hours and most preferably of at least forty-eight hours.
The phrase xe2x80x9cpurified TAFIaxe2x80x9d means a TAFIa composition that has TAFIa as predominantly a single-band by coomassie-stained, or more preferably, silver-stained, SDS-PAGE gel. Preferably, the TAFIa is a single band on a coomassie-stained, or more preferably, silver-stained, SDS-PAGE gel. Preferably, the purified TAFIa is substantially free from thrombin-thrombomodulin. Preferably, TAFIa has been purified after activation by a purification step such as including, but not limited to, size-exclusion chromatography, affinity chromatography or ammonium sulfate precipitation. In a preferred embodiment, TAFIa is purified by affinity chromatography on a potato tuber carboxypeptidase inhibitor (PTCI)-Sepharose column. In specific embodiments, the purified TAFIa ia at least 75%, 80%, 85%, 90%, 95%, 99% or preferably at least 99.5% of the composition.
The term xe2x80x9cprophylactic agentxe2x80x9d means an agent capable of preventing or reducing the risks or incidence of a disease or disorder in a patient. In one embodiment, the prophylactic agent is one which is capable of preventing a hemorrhagic or thrombotic disorder.
The phrase xe2x80x9cprophylactically effective amountxe2x80x9d refers to that amount of the prophylactic agent sufficient to result in the prevention or reduction in the risk or incidence of the hemorrhagic or thrombotic disorder. Further, a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or in combination with other agents, that provides a prophylactic benefit in the prevention of the hemorrhagic or thrombotic disorder.
The term xe2x80x9ctherapeutic agentxe2x80x9d means an agent capable of modifying, controlling, delaying or reversing a disease or disorder or ameliorating the symptoms of a disease or disorder in a patient. In one embodiment, the therapeutic agent is one which is capable of modifying, controlling, delaying, reversing or ameliorating the symptoms of a hemorrhagic or thrombotic disorder.
The phrase xe2x80x9ctherapeutically effective amountxe2x80x9d refers to that amount of the therapeutic agent sufficient to modify, control, reverse or ameliorate the symptoms of the hemorrhagic or thrombotic disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay, minimize, reverse or ameliorate the symptoms of the hemorrhagic or thrombotic disorder. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of the hemorrhagic or thrombotic disorder. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of the hemorrhagic or thrombotic disorder, including the amelioration of symptoms associated with the hemorrhagic or thrombotic disorder being treated.
The phrase xe2x80x9cin combinationxe2x80x9d refers to the use of more than one prophylactic and/or therapeutic agents against a hemorrhagic or thrombotic disease or disorder.
The term xe2x80x9cpatientxe2x80x9d refers to a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, mice, rats etc.) or a primate (e.g., monkeys and humans). Preferably the patient is a human.
The terms xe2x80x9cmanagexe2x80x9d, xe2x80x9cmanagingxe2x80x9d and xe2x80x9cmanagementxe2x80x9d refer to the beneficial effects that a patient derives from a prophylactic or therapeutic agent, which does not result in a cure of the disorder. In certain embodiments, a patient is administered one or more prophylactic or therapeutic agents to xe2x80x9cmanagexe2x80x9d a disease or disorder, or symptoms associated with the disease or disorder, so as to prevent the progression or worsening of the disease or disorder.
The terms xe2x80x9cpreventxe2x80x9d, xe2x80x9cpreventingxe2x80x9d and xe2x80x9cpreventionxe2x80x9d refer to the prevention of the recurrence, spread or onset of the hemorrhagic or thrombotic disease or disorder, or symptoms associated with the disease or disorder, in a patient resulting from the administration of a prophylactic or therapeutic agent.
The terms xe2x80x9ctreatxe2x80x9d, xe2x80x9ctreatingxe2x80x9d and xe2x80x9ctreatmentxe2x80x9d refer to the, modification, control, reversal or amelioration of the symptoms of the hemorrhagic or thrombotic disease or disorder that results from the administration of one or more prophylactic or therapeutic agents. In certain embodiments, such terms refer to the minimizing or delay of the hemorrhagic or thrombotic disease or disorder resulting from the administration of one or more prophylactic or therapeutic agents to a patient with such a disease or disorder.
The term xe2x80x9ct-PAxe2x80x9d refers to native t-PA and recombinant t-PA as well as homologs, derivatives and polymorphisms of t-PA that retain the catalytic activity of native t-PA.